In Vivo Optical Properties of Melanocytic Skin Lesions: Common Nevi, Dysplastic Nevi and Malignant Melanoma

We present an in vivo study of the optical properties of common nevi, dysplastic nevi and malignant melanoma skin lesions in human subjects. Reflectance spectra were measured on 1379 skin lesions, in the visible and near-infrared spectral regions, using a spectral imaging system, in a clinical setting. Analysis of the data using a reflectance model revealed differences between the optical properties of melanin present in nevi and melanoma lesions. These differences, which are in agreement with our previous observations on average reflectance spectra, may be potentially useful for the noninvasive characterization of pigmented skin lesions and the early diagnosis of melanoma.     

Monte Carlo simulation of near infrared autofluorescence measurements of in vivo skin

The autofluorescence properties of normal human skin in the near-infrared (NIR) spectral range were studied using Monte Carlo simulation. The light-tissue interactions including scattering, absorption and anisotropy propagation of the regenerated autofluorescence photons in the skin tissue were taken into account in the theoretical modeling. Skin was represented as a turbid seven-layered medium. To facilitate the simulation, ex vivo NIR autofluorescence spectra and images from different skin layers were measured from frozen skin vertical sections to define the intrinsic fluorescence properties. Monte Carlo simulation was then used to study how the intrinsic fluorescence spectra were distorted by the tissue reabsorption and scattering during in vivo measurements. We found that the reconstructed model skin spectra were in good agreement with the measured in vivo skin spectra from the same anatomical site as the ex vivo tissue sections, demonstrating the usefulness of this modeling. We also found that difference exists over the melanin fluorescent wavelength range (880-910 nm) between the simulated spectrum and the measured in vivo skin spectrum from a different anatomical site. This difference suggests that melanin contents may affect in vivo skin autofluorescence properties, which deserves further investigation.     

Absorption spectra of human skin in vivo in the ultraviolet wavelength range measured by optoacoustics

Knowledge of the optical properties of human skin in the ultraviolet range is fundamental for photobiologic research. However, optical properties of human skin in the ultraviolet spectral range have so far mainly been measured ex vivo . We have determined the absorption spectra of human skin in vivo in the wavelength range from 290 to 341 nm in 3 nm steps using laser optoacoustics. In this technique, optical properties are derived from the pressure profile generated by absorbed light energy in the sample. In a study on 20 subjects belonging to phototypes I–IV, we studied the optical properties at the volar and dorsal aspect of the forearm as well as on the thenar. Analysis of the measured absorption spectra shows that comparable skin areas—like different sides of the forearm—have qualitatively similar optical characteristics. Still, the optical properties may vary substantially within the same area, probably due to the skin structure and inhomogeneities. Comparison of the spectra from different skin sites indicates that the spectral characteristics of the stratum corneum and its chromophores play an important role for the optical properties of human skin in vivo in the ultraviolet B range.     

Probing near-infrared photorelaxation pathways in eumelanins and pheomelanins

Ultraviolet-visible spectroscopy readily discerns the two types of melanin pigments (eumelanin and pheomelanin), although fundamental details regarding the optical properties and pigment heterogeneity are more difficult to disentangle via analysis of the broad featureless absorption spectrum alone. We employed nonlinear transient absorption spectroscopy to study different melanin pigments at near-infrared wavelengths. Excited-state absorption, ground-state depletion, and stimulated emission signal contributions were distinguished for natural and synthetic eumelanins and pheomelanins. A starker contrast among the pigments is observed in the nonlinear excitation regime because they all exhibit distinct transient absorptive amplitudes, phase shifts, and nonexponential population dynamics spanning the femtosecond-nanosecond range. In this manner, different pigments within the pheomelanin subclass were distinguished in synthetic and human hair samples. These results highlight the potential of nonlinear spectroscopies to deliver an in situ analysis of natural melanins in tissue that are otherwise difficult to extract and purify.     

Monte Carlo simulation of cutaneous reflectance and fluorescence measurements--the effect of melanin contents and localization

Melanin content and distribution in skin were studied by examining a patient with white, brown and blue skin tones expressed on skin affected by vitiligo. Both diffuse reflectance and autofluorescence spectra of the three distinction skin sites were measured and compared. Monte Carlo simulations were then performed to help explain the measured spectral differences. The modeling is based on a six-layer skin optical model established from published skin optical parameters and by adding melanin content into different locations in the model skin. Both the reflectance and fluorescence spectra calculated by Monte Carlo (MC) simulation were approximately in agreement with experimental results. The study suggests that: (1) trichrome vitiligo skin may be an ideal in vivo model for studying the effect of skin melanin content and distribution on skin spectroscopy properties. (2) Based on the skin optical model and MC simulation, the content and distribution of melanin in skin, or other component of skin could be simulated and predicted. (3) Both reflectance and fluorescence spectra provided information about superficial skin structures but fluorescence spectra are capable of providing information from deeper cutaneous structures. (4) The research method, including the spectral ratio method, the method of adding and modifying the melanin content in skin optical models, and MC simulation could be applied in other non-invasive optical studies of the skin.     

The action spectrum for generation of the primary intermediate revealed by ultrafast absorption spectroscopy studies of pheomelanin

The observation that fair-skinned individuals are more susceptible to skin cancers is commonly explained by invoking an enhanced photoreactivity of the red melanin, pheomelanin compared with the black melanin, eumelanin. For the wavelength range from 500 to 1000 nm, pump-probe spectroscopic measurements reveal the photoexcitation of pheomelanin by UVA light that generates an immediate (< 100 fs) transient absorption centered at 780 nm. Using a tunable femtosecond excitation source, the action spectrum between 300 and 390 nm for generation of the primary intermediate was measured. Similar action spectra are found for the sample with molecular weight (MW) between 1000 and 10 000 and the one with MW > 10 000 fractions of pheomelanin, indicating that the reactive chromophore has a low MW but is present and its photophysics is similar in the aggregated pigment. The shape of the action spectrum differs from the absorption spectrum of bulk melanin and mass-selected fractions but resembles reported absorption spectrum of benzothiazines, oxidation products of 5-S-cysteinyl-dopa, which are formed along the biosynthetic pathway of pheomelanin.     

On the assessment of melanin in human skin in vivo

Abstract— A method for estimating the amount of pigment in normal human skin in uivo is presented. This method is based on remittance spectroscopy. The spectrum of normal skin is compared to amelanotic skin and the logarithm of the ratio is fitted with a straight line in the range 620-720 nm. The parameters obtained are strongly correlated for all the volunteers in this study. therefore each spectrum determines one parameter for each individual. When similar analysis was performed on DOPA-melanin we obtained the same strong correlation among different concentrations. We are thus able to determine a relation between the coefficient obtained from the remittance spectrum from normal skin and an equivalent concentration of DOPA-melanin in aqueous solution. We can thus estimate. to a first approximation, the total melanin mass in human skin non-invasively, and can determine a parameter that is uniquely correlated to the amount of melanin pigment in the skin.     

The melanosome: threshold temperature for explosive vaporization and internal absorption coefficient during pulsed laser irradiation

The explosive vaporization of melanosomes in situ in skin during pulsed laser irradiation (pulse duration less than 1 microsecond) is observed as a visible whitening of the superficial epidermal layer due to stratum corneum disruption. In this study, the ruby laser (694 nm) was used to determine the threshold radiant exposure, H0 (J/cm2), required to elicit whitening for in vitro black (Negroid) human skin samples which were pre-equilibrated at an initial temperature, Ti, of 0, 20, or 50 degrees C. A plot of H0 vs Ti yields a straight line whose x-intercept indicates the threshold temperature of explosive vaporization to be 112 +/- 7 degrees C (SD, N = 3). The slope, delta H0/delta Ti, specifies the internal absorption coefficient, mua, within the melanosome: mua = -rho C/(slope(1 + 7.1 Rd)), where rho C is the product of density and specific heat, and Rd is the total diffuse reflectance from the skin. A summary of the absorption spectrum (mua) for the melanosome interior (351-1064 nm) is presented based on H0 data from this study and the literature. The in vivo absorption spectrum (380-820 nm) for human epidermal melanin was measured by an optical fiber spectrophotometer and is compared with the melanosome spectrum.     

Determination of wavelength-specific UV protection factors of sunscreens in intact skin by EPR measurement of UV-induced reactive melanin radical

There remains an unmet need for skin tissue-based assays for the measurement of the UVA protection and efficacy of sunscreens. Here we describe development of a novel electron paramagnetic resonance assay that uses the photogeneration of reactive melanin radical as a measure of UV light penetration to melanocytes in situ in skin. We have used areas of focal melanocytic hyperplasia in the skin of Monodelphis domestica to model the human nevus. We show that we are able to use this assay to determine the monochromatic protection factors (mPF) of research and commercial sunscreens at specific narrow wavebands of UVB, UVA and blue visible light. Both commercial sunscreens, a sun protection factor (SPF) 4 and an SPF 30 product, had mPFs in the UVB range that correlated well with their claimed SPF. However, their mPF in the UVA ranges were only about one-third of claimed SPF. This technique can be used to design and assay sunscreens with optimally balanced UVA and UVB protection.     

OPTICAL AND THERMAL CHARACTERIZATION OF NATURAL (Sepia officinalis) MELANIN

Abstract The optical properties and the thermal diffusivity of natural cuttlefish ( Sepia officinalis ) melanin have been measured. The optical absorption and scattering properties of melanin particles were determined at 580 nm and 633 nm, using photometric and photothermal techniques. For the photometric studies, the absorption and the transport scattering coefficients were determined from the measurements of diffuse reflectance and transmittance. The scattering anisotropy was obtained from an additional measurement of the total attenuation coefficient and independently obtained by goniometry. For photothermal studies, pulsed photothermal radiometry was used to deduce the absorption and transport scattering coefficients via a model based on optical diffusion theory. Pulsed photothermal radiometry was also used to provide the thermal diffusivity of solid melanin pressed pellets.     

Optical properties of in vitro epidermis and their possible relationship with optical properties of in vivo skin.

Using a spectrophotometer with an internal integrating sphere, the absorption (mu a) and reduced scattering (mu s') coefficients of in vitro epidermis were evaluated from reflectance and transmittance measurements. mu a and mu s' varied from 24 to 0.2 cm-1, and from 32 to 21 cm-1 respectively, on passing from 400 to 800 nm. Moreover, using an external integrating sphere, the reflectance spectrum of in vivo skin was compared with the reflectance spectrum calculated with a Monte Carlo model, in which the mean values of mu a and mu s' and different anisotropy parameters were used as input data. In vivo results show that the principle of similarity is entirely valid for wavelengths greater than 600 nm and may be considered a good approximation in the 400-600 nm band, and suggest that optical characteristics of in vivo skin may be inferred from reflectance measurements.     

Modeling and verification of melanin concentration on human skin type

Lasers are used in the minimalistic or noninvasive diagnosis and treatment of skin disorders. Less laser light reaches the deeper skin layers in dark skin types, due to its higher epidermal melanin concentration compared with lighter skin. Laser–tissue interaction modeling software can correct for this by adapting the dose applied to the skin. This necessitates an easy and reliable method to determine the skin’s type. Noninvasive measurement of the skin’s melanin content is the best method. However, access to samples of all skin types is often limited and skin‐like phantoms are used instead. This study’s objective is to compare experimentally measured absorption features of liquid skin‐like phantoms representing Skin Types I–VI with a realistic skin computational model component of ASAP^®. Sample UV–VIS transmittance spectra were measured from 370 to 900 nm and compared with simulated results from ASAP^® using the same optical parameters. Results indicated nonmonotonic absorption features towards shorter wavelengths, which may allow for more accurate ways of determining melanin concentration and expected absorption through the epidermal layer. This suggests possible use in representing optical characteristics of real skin. However, a more comprehensive model and phantoms are necessary to account for the effects of sun exposure.     

Ultrafast spectral hole burning reveals the distinct chromophores in eumelanin and their common photoresponse

Eumelanin, the brown-black pigment found in organisms from bacteria to humans, dissipates solar energy and prevents photochemical damage. While the structure of eumelanin is unclear, it is thought to consist of an extremely heterogeneous collection of chromophores that absorb from the UV to the infrared, additively producing its remarkably broad absorption spectrum. However, the chromophores responsible for absorption by eumelanin and their excited state decay pathways remain highly uncertain. Using femtosecond broadband transient absorption spectroscopy, we address the excited state behavior of chromophore subsets that make up a synthetic eumelanin, DOPA melanin, and probe the heterogeneity of its chromophores. Tuning the excitation light over more than an octave from the UV to the visible and probing with the broadest spectral window used to study any form of melanin to date enable the detection of spectral holes with a linewidth of 0.6 eV that track the excitation wavelength. Transient spectral hole burning is a manifestation of extreme chemical heterogeneity, yet exciting these diverse chromophores unexpectedly produces a common photoinduced absorption spectrum and similar kinetics. This common photoresponse is assigned to the ultrafast formation of immobile charge transfer excitons that decay locally and that are formed among graphene-like chromophores in less than 200 fs. Raman spectroscopy reveals that chromophore heterogeneity in DOPA melanin arises from different sized domains of sp²-hybridized carbon and nitrogen atoms. Furthermore, we identify for the first time striking parallels between the excited state dynamics of eumelanin and disordered carbon nanomaterials, suggesting that they share common structural attributes.

Seeing the colors in black: ultrafast transient hole burning spectroscopy reveals the absorption properties of discrete chromophores and their interactions in the skin pigment eumelanin.     

Mapping the Distribution of Melanin Concentration in Different Fitzpatrick Skin Types Using Hyperspectral Imaging Technique

Measuring skin melanin concentration in order to assess skin phototype according to Fitzpatrick's classification is a constant research goal. In this study, a new approach for assessing skin melanin concentration based on hyperspectral imaging combined with an appropriate analytical model that exploits specific spectral bands to generate maps of melanin content distribution on different Fitzpatrick skin phototypes is presented. Hyperspectral images from the proximal inner side of the forearms of 51 young volunteers covering the first four classes of Fitzpatrick's phototypes were acquired using a hyperspectral imaging system. The images were analyzed using a modified Beer–Lambert law that segregates the contribution of melanin from the other constituents to the skin absorption spectrum. The performance of the model was evaluated using the coefficient of determination (r-squared). The results revealed that the approach proposed in this study generated accurate melanin concentration distribution maps that allowed a correct classification of skin phototype. In conclusion, the proposed approach for assessing skin melanin concentration proved to be very reliable for classifying skin phototypes, and, as it provides maps that are easily read, it has the advantage of a possible extension of its applications to other research concerning skin pigmentation.     

Increased melanogenesis is a risk factor for oxidative DNA damage--study on cultured melanocytes and atypical nevus cells

Melanin synthesis is an oxygen-dependent process that acts as a potential source of reactive oxygen species (ROS) inside pigment-forming cells. The synthesis of the lighter variant of melanin, pheomelanin, consumes cysteine and this may limit the capacity of the cellular antioxidative defense. We show that tyrosine-induced melanogenesis in cultured normal human melanocytes (NHM) is accompanied by increased production of ROS and decreased concentration of intracellular glutathione. Clinical atypical (dysplastic) nevi (DN) regularly contain more melanin than do normal melanocytes (MC). We also show that in these cultured DN cells three out of four exhibit elevated synthesis of pheomelanin and this is accompanied by their early senescence. By using various redox-sensitive molecular probes, we demonstrate that cultured DN cells produce significantly more ROS than do normal MC from the same donor. Our experiments employing single-cell gel electrophoresis (comet assay) usually reveal higher fragmentation of DNA in DN cells than in normal MC. Even if in some cases the normal alkaline comet assay shows no differences in DNA fragmentation between DN cells and normal MC, the use of the comet assay with formamidopyrimidine DNA glycosylase can disclose that the DNA of the cultured DN cells harbor more oxidative damage than the DNA of normal MC from the same person.     

Amount of Melanin Granules in Human Hair Defines the Absorption and Conversion to Heat of Light Energy in the Visible Spectrum

One of the known important functions of hair is protection from extensive sunlight. This protection is accomplished in large part due to natural hair pigmentation which is known to reflect the number of melanin granules (melanosomes) in the hair shaft, and melanin variants. Melanin takes in excessive light energy and converts it to heat in a process called absorption; heat is then dissipated into the environment as infrared radiation, thereby protecting the underlying skin. We used transmission electron microscopy (TEM) to visualize the melanosome counts in samples of human hair, and used thermal microscopy to measure the temperature changes of the samples when exposed to green and blue light lasers. In our experiments green light conversion to heat was highly correlated to the number of melanosomes, whereas blue light conversion to heat was less correlated, which may be because the reddish melanosomes it contains are less effective in absorbing energy from the blue spectrum of light. Anyway, we have shown the metals accumulation in the melanin can be easily visualized with TEM. We confirmed that the amount of melanin granules in human hair defines the conversion to heat of light energy in the visible spectrum.     

In vivo SPECTROPHOTOMETRIC EVALUATION OF NEOPLASTIC AND NON-NEOPLASTIC SKIN PIGMENTED LESIONS–I. REFLECTANCE MEASUREMENTS

Reflectance spectrophotometry from 400 to 800 nm on different cutaneous pigmented lesions, including primary and metastatic malignant melanoma, pigmented nevi, lentigo and seborrhoeic keratosis, has been performed by using an external integrating sphere coupled to a spectrophotometer. Measurements show that reflectance spectra of the different lesions manifest dissimilar patterns, particularly in the near IR region. Comparison of reflectance of nevi with that of malignant melanomas results in a highly significant difference (P less than 10(-6)) between the two samples. Though interpretation of the spectra remains difficult as a result of the complexity of the optical processes of scattering and absorption, our results suggest that a detailed analysis of the reflectance spectrum may give clinically useful information, and could be utilized as an aid in clinical diagnosis of cutaneous pigmented lesions, especially where malignant melanoma is concerned.     

Electronic excitations in many-particle systems: a quantitative analysis

Small particles, produced in usual experiments, commonly form many-particle systems. Interactions of various kinds among the particles influence the properties of such systems. We consider here the optical properties of noble metal particle systems with separated nearly spherical particles and with aggregates formed by induced coagulation of the single particles. In order to describe their optical extinction we apply electrodynamic interaction models on particles and aggregates. We perform a quantitative analysis of the extinction spectrum of one selected sample with the electrodynamic interaction model.     

The importance of the depth distribution of melanin in skin for DNA protection and other photobiological processes

Melanin pigments are important regulators for the evolution of essential functions of human skin. The concentration of melanin, as well as its depth distribution, is strongly affected by ultraviolet radiation. In un-tanned skin, melanin pigments are found only in the basal layer of the epidermis, while in tanned skin it is distributed throughout the epidermis. So far, mainly the amount of melanin, and not its distribution, has been considered in view of skin photobiology. With an advanced radiative transfer model we investigate, for the first time, how the depth distribution of melanin influences the amount of ultraviolet radiation that reaches living cells in the epidermis, and thus can damage the DNA in the cells. The simulations are performed for average pigmented skins (type III-IV). A surprisingly large factor, up to 12, is found between the ultraviolet protection of skin with melanin distributed throughout the epidermis, and skin with melanin only in the basal layer of the epidermis. We also show that the synthesis of previtamin D3, in skin, can vary with more than 100% if the depth distribution of melanin is changed, while the degradation of folate in dermal blood is almost un-affected by variations in the melanin depth distribution.     

Role of UV in cutaneous melanoma

Malignant melanoma arises from epidermal melanocytes, the cells responsible for the production of the skin pigment melanin. The photoprotective role of melanin, which is transferred to neighboring keratinocytes, in UV-induced skin carcinogenesis, specifically in nonmelanoma skin cancers, has been well documented. Although melanocyte-resident melanin is expected to offer similar protection to melanocytes from UV-induced damage, UV radiation has long been suspected to have an etiologic role in cutaneous melanoma. However, nearly three decades of efforts using a variety of in vitro and in vivo models of human skin and mouse genetic models have produced conflicting data. Epidemiologic studies have also failed to establish a definitive association between UV exposure and risk of melanoma. In this review, we evaluate the dual role of the melanin pigment as a photoprotector as well as a photosensitizer and examine the evidence for association between melanin levels (constitutive and induced) and melanoma risk. We also discuss possible reasons for the lack of signature UV mutations in melanoma oncogenes known to date and potential alternative mechanisms to explain the role of UV in melanomagenesis.